Microtubules and force-producing enzymes termed "motor proteins" power chromosome segregation during cell division, ciliary beating, and the rapid transport of membranous organelles in the endocytic and secretory pathways. Deciphering the molecular basis of microtubule-based motility is therefore germane to understanding many intracellular processes that are needed for the growth, survival and development of eukaryotic cells. Furthermore, mutations in kinesin and dynein motors often produce defects in ciliary movement, meiosis, mitosis and axonal transport in many organisms; similar mutations may underlie certain human diseases as well. The long-term objectives of this laboratory are to elucidate the biological roles and biophysical properties of kinesin and dynein motors. One aim of this grant is to understand chemomechanical transduction, the process by which motors move along microtubules. In vitro motility assays and high resolution microscopy capable of detecting motion with nanometer precision on a millisecond time scale will be used to measure the discrete mechanical actions of individual motor proteins. By relating such mechanical events to chemical intermediates in the ATP hydrolysis cycle using ATP analogues, a general mechanism for the force- generating process may be defined. A second aim of this grant is to identify the proteins that are needed for initiating and regulating organelle transport along microtubules. Reconstituted organelle transport assays developed in this laboratory will be used in combination with classical biochemical techniques to purify proteins that are needed in conjunction with kinesin and dynein to move organelles in a specific direction. The long range goal of this effort is to derive a completely defined organelle transport system using purified components, so that the transport machinery can be dissected at a molecular level. Our third objective is to define the biological roles of kinesins in living cells by genetic approaches as well as by immunocytochemical localization of the motors. Several kinesin genes in Dictyostelium discoideum, a unicellular motile slime mold, will be disrupted by homologous recombination, and the phenotype of the mutant cell will be examined. Through such studies, biological functions can be assigned to specific kinesin motors. The roles of two human conventional kinesin heavy chains, one of which is ubiquitously expressed and the other of which is brain specific, will be examined by determining their respective cellular and subcellular distributions and by disrupting their function by antibody microinjection or anti-sense oligonucleotide treatment in cell culture systems.